Hydroxylamino acetic acid derivatives



Uflitfid S te Paten 3,202,760 HYDRQXYLAMENO ACETIC ACID DERIVATIVES Edward A. Kaczka, Union, and Eugene L. Dulaney, Me-

tnchen, Ni, assignors to Merck & Co., Inc., Rahway, NJ., a corporation of New Jersey N0 Drawing. Filed Apr. 29, 1960, Ser. No. 25,506 Claims. (Cl. 260-482) of the objects of this invention, may be represented in the free acid form by the general formula:

Rl-ITT CHzC O O H Where R is an acyl radical. Also included Within the invention are the salts and esters of such N-acyl hydroxylamino acetic acids, so that the generic structural formula may be pictured as:

o it Ri-NCHz 0Ra 0R2 where R is' an acyl radical, R may be hydrogen or a metal, and R may be hydrogen, a metal or a hydrocarbon group. it;

The acyl radical represented by R in the above for-- mula may be a forrnyl group, or another lower alkanoyl radical such as acetyl, haloacetyl, propionyl, butyryl or valeroyl, an aroyl group such as benzoyl, an aralkanoyl radical of the type represented by phenylacetyl, and the residue of a heterocyclic acid such as a fur'oyl or thienoyl radical. It is preferred that the R group contain less than nine carbon atoms, and the compounds where R is a formyl radical are the preferred species of "the invention.

In addition to the free acids (where R and R are hydrogen), the alkalimetal and alkaline earth metal salts, such as the sodium, potassium, lithium, calcium and barium salts, and thelower alkyl esters exemplified by methyl, ethyl, propyl and isopropyl esters, represent the preferred embodiments of R and R The N-acyl hy'droxylamino acetic acids described herein" may be prepared by treatment of hydroxylamino acetic acid or a salt thereof with a suitable N-acylating" agent. An acyl halide, such as an acyl chloride or bromide, is generally satisfactory for this purpose. The reaction is preferably carried out by intimately contacting the hydroxylamino acetic acid with a slight molar'excess of acyl halide at temperatures of 35 C. or less The acylation proceeds rapidly, and is normally substantially complete in'less than about 1 hour, although longer times and/ or higher temperatures may be used if desired. It is convenient to employ an excess of the acylating agent as reaction solvent, but cold dilute aqueous alkali may also be used. In the reaction with an acyl halide, both N- and O-acylation may take place, particularly in an anhydrous However, heavy: metal salts and other esters such as the benzyl, phenethyl, allyl and like esters may be produced accordingjto the in the carbon and nitrogen sources.

3,202,7d0 Patented Aug. 24, 1965 7 ice ylamino acetic acid compounds give a positive ferric chloride test whereas the O-acyl derivatives do not.

In this manner, compounds such as N-acetyl, N-benzoyl, N-phenylacetyl, 'N-propionyl, N-furoyl, N-butyryl and N-chloracetyl hydroxyamino acetic acids are obtained from the appropriate N-acyl halide and'hydroxylamino acetic acid.

Alternatively, the N-acyl hydroxylaminoacetic acids, where the acyl radical is the residue of a lower fatty acid, are conveniently made by contacting hydroxylamino acetic acid with the lower aliphatic acid, preferably in the presence of an' acid anhydride. In this way, N-formyl hydroxylamino acetic acid is obtained from formic acid and hydroxylamino acetic acid. The reaction is carried out at about room temperature, and is substantially complete in 2 hours or less; It will be appreciated by those skilled in this art that salts of the N-acyl hydroxylamino acetic acids will be obtained from the reaction mixture rather than the free acid if the pH is 7 or above.

In accordance with an additional aspect of this invention, it has been discovered that N-formyl hydroxylamino acetic acid may also be produced in good yields by fermentation of a nutrient medium with a suitable microrescens spe'cies of the Penicilliumfrequentans series, and

of the Penicillium aurantio-violaceum species of the Penicillium lividum series.

In order to prepare. N-formyl hydroxylamino acetic acid by this fermentation method, a producing strain of Penicillium of the type described above is grown under submerged aerated conditions'in a nutrient medium containing an assimilable source of carbon and nitrogen. The particular nutrient medium is not critical. Suitable carbon sources include a carbohydratesuch as dextrose, sucrose, starch, glycerol or molasses, whereas suitable sources of nitrogeninclude cornsteep liquor, yeast extract, soybean meal, cotton seed meal, casein, distillers solu-' bles and the like. Thefermentation medium also contains trace amounts of salts which are normally contained The nutrientmedium is normally adjusted to a pH in the range of 6-8, and sterilized before inoculation Withthe producing strain of Penicillium. Substantial quantities of N -formyl hydroxylaminoacetic acid are normally produced in the fermentation medium after a growth period'of approximately-4'10 days at temperatures in the range of 25 30 C. With the preferred microorganism, about 2-15 grams of N-formyl hydroxylamino acetic acid per liter of fermentation broth is produced under these conditions.

In order to recover the N-formyl hydroxylamino acetic acid from the fermentation broth, the mycelia are. first removed by techniques such as filtrationor centrifugation. The filtered broth is concentrated essentially to dryness by techniques known in this art. The solid residue is then extracted with methanol and the methanol extracts, which contain the desired product, are concentrated to dryness or near dryness. Final purification is achieved by crystallization from aqueous ethanol. This method of recovering N-formyl hydroxylamino acetic acid may be employed to obtain either the free acid or an alkali metal salt thereof. Unless pH adjustments are made at conclusion of fermentation or during the isolation process, the product obtained will depend upon the pH of the finished fermentation broth. It is normally preferred to start the fermentation at a pH of 6-8.

If it is desired, N-formyl hydroxylamino acetic acid may be synthesized by bringing the producing enzyme system elaborated by the above-described microorganisms into intimate contact with an aqueous medium containing sources of carbon and nitrogen. With this procedure, it is not necessary or contemplated that a growing culture of the Penicillium be used.

The N-acyl hydroxylamino acetic acids, and their salts and esters, exhibit absorption in the infra-red portion of the spectrum, give a red-purple color with ferric chloride, do not reduce Fehlings solution, decolorize permanganate, give a yellow color with bromphenol blue and an atypical ninhydrin test. They may be hydrolyzed with acid to hydroxylamino acetic acid and to the organic acid corresponding to the N-acyl radical.

It has been found convenient, particularly in the case of the N-formyl hydroxylamino acetic acid when prepared by fermentation, to recover the product as an alkali metal salt such as the sodium salt. The salts have the activity of the parent acid and may be employed as such or converted to the acid by treatment with an aqueous mineral acid or by ion exchange resin metathesis. N-formyl hydroxylamino acetic acid exists in two crystalline forms, cubes melting at 108-110 C. and needles melting at 119120 C.

Salts of the N-acyl hydroxylamino acetic acids described herein are readily obtained by treating a solution of acid with an appropriate base such as ammonium hydroxide, potassium hydroxide, sodium carbonate, calcium carbonate, lithium hydroxide and the like. It will be realized that monoand di-salts can be produced depending on the amount of base employed, the di-salts forming at a pH or about 10 and above.

Similarly, esters are synthesized by treatment of the N-acylated acids with a suitable esterifying agent such as a diazoalkane. In this manner, lower alkyl esters such as the methyl, ethyl and propyl esters of N-acyl hydroxylamino acetic acids are obtained, which compounds possess the activity of the acids and salts discussed in more detail hereinbelow. Alternatively, esters of N-acyl hydroxylamino acetic acid may be obtained by transesterification of the methyl ester with an appropriate alcohol, or by esterification of hydroxylamino acetic acid and subsequent N-acylation of the ester. This latter method is convenient since the esterification may be acid catalyzed without risk of hydrolyzing the N-acyl group.

The monosodium salt of N-formyl hydroxylamino acetic acid, which is the species normally recovered from a fermentation broth, exists as a hydrate as well as in anhydrous form as shown by infra-red spectra determinations. The spectra can be divided into two categories: (a) those which show free hydroxyl absorption (band at 2.8 1), and (b) those in which this band is absent. The anhydrous material has characteristic infra-red absorption at the longer Wavelengths which is readily distinguishable from the hydrate.

The characteristic peaks of absorption in the infra-red region of the spectrum of the two forms of sodium N-formyl hydroxylamino acetate, as well as the absorption peaks of N-formyl hydroxylamino acetic acid, and the ammonium, potassium and calcium salts thereof are at the following wavelengths in microns:

g Sodium N-formyl hydroxylamino acetate (anhydrous): 2.8-4.Z u (broad), 5.9, 6.3, 6.6, 7.0, 7.6, 8.3, 8.4, 9.8, 10.1,11.2,13.0.

Sodium N-formyl hydroxylamino acetate (hydrate): 2.82.9, 3.7-4.2 (broad), 6.0, 6.3, 6.6, 7.5, 8.2, 10,1. 11.2, 12.8.

Disodium N-formyl hydroxylamino acetate: 2.9, 3.1, 6.1, 6.3, 7.6, 8.2, 8.3, 10.2, 10.4, 10.7, 11.0, 11.7-12.0, 12.8.

N-formyl hydroxylamino acetic acid: 2.8-4.5 (broad), 5.8, 6.1, 7.2, 7.5, 8.0, 8.2, 8.4, 10.2, 10.8, 11.1, 13.0.

Ammonium N-formyl hydroxylamino acetate: 3.1-5.0 (broad), 5.9-6.1, 6.3, 6.5, 7.7, 8.3, 10.2, 10.8, 11.3, 12.9.

Potassium N-formyl hydroxylamino acetate: 3.0-4.7 (broad), 6.0, 6.5, 7.6, 8.2, 8.4, 9.9, 10.2, 11.2, 13.0.

Calcium N-formyl hydroxylamino acetate: 3.1, 5.9, 6.1, 6.3, 7.2, 7.6, 8.3, 10.1, 10.7, 10.9.

The spectra were taken on a Perkin-Elmer Model 137 Infra-red Spectophotometer (Infracord) on a sample suspended in mineral oil (Nujol).

The compounds of this invention are highly effective in killing or inhibiting the growth of certain types of plants. They are particularly useful in that they kill or inhibit growth of weeds and other undesirable plants such as crab grass, dandelions, sheep sorrel, plantains and wild brush. The treatment is normally carried out by spraying a solution of the N-acyl hydroxylamino acetic acid (or salt or ester derivative thereof) on the plants, preferably to run-off. Effective results are obtained with small amounts of active ingredient, i.e., with solutions containing from 30-1000 p.p.m. of N-acyl hydroxylaminov acetic acid.

In addition to using solutions of pure or substantially pure compounds for this purpose, highly satisfactory results are obtained with concentrates of the active materials. This is of particular significance in the case of N-formyl hydroxylamino acetic acid compounds because it permits the use of crude fermentation concentrates, and even of the fermentation broth itself, for control of weeds and other'undesirable plants. The N-acyl hydroxylamino acetic acids described herein likewise inhibit the growth of Alaska pea plants, germinated tomato seeds and Pinto bean plants. This property may be conveniently used as a means of determining the presence and concentration of our new compounds in a material, or as a method of standardizing preparations thereof.

The following examples are given for the purpose of illustration and not by way of limitation:

Example 1 A. 355 mg. of hydroxylamino acetic acid are added to a mixture of 5 ml. of 98% formic acid and 1 ml. of acetic anhydride. The resulting mixture is stirred for 15 minutes at a temperature of 25 C. The solution is then heated at 5055 C. for 10 minutes, and then evaporated to dryness in vacuo at a temperature of about 25 C. The resulting residue is dissolved in 2 ml. of water and the .pH of the solution adjusted to between 7 and 8 with dilute aqueous sodium hydroxide. The resulting solution is then concentrated to a volume of about 3 ml. and 30 ml. of ethanol added to it. Crystals of sodium N-formyl hydroxylamino acetate precipitate and are recovered by filtration. They are dissolved in 4 ml. of water and 20 ml. of ethanol are added to the resulting solution. The resulting suspension is filtered to give 357 mg. of crystalline sodium N-formyl hydroxylamino acetate, M.P. 205 -210 C. (dec.).

B. When 2 ml. of 98% formic acid and 63 mg. of hydroxylarnino acetic acid are reacted together for 15 minutes at 25 C., and the reaction mixture then concentrated to dryness, N-forrnyl hydroxylamino acetic acid is obtained.

Example 2 216 mg. of hydroxylamino acetic acid is dissolved in 3 ml. of acetic anhydride and the solution allowed to stand at about 25 C. for about 16 hours. The solution is then evaporated to dryness in vacuo to yield a crystalline residue. These crystals are dissolved in acetone and to give N-acetyl hydroxylamino acetic acid.)

106 mg. of N-acetyl-N-acetoxyam-ino acetic acid is dissolved in about 3 ml. of water and the solution allowed to stand at about room temperature for 16 hours. The solution isthen'evaporated to dryness in vacuo. The residue is dissolved in about 0.5 ml. of water and the pH adjusted to 7-8 with dilute sodium hydroxide. This solution is evaporated to near dryness and the residue tr-iturated with ethanol which induces crystallization of the monosodium salt of N-acetyl hydroxylamin-o acetic acid.

The infra-red absorption spectrum of a substantially pure sample of the'monosodium salt of N-acetyl hydroxylamino acetic acid suspended in mineral oil (Nujol) taken on a Perkin-Elmer Model 137 Infrared 'Spectro photometer (Infracord) shows characteristic peaks at the following wavelengths,*expressed in microns: 6.1, 6.3, 6.6, 7.75, 8.2, 9.35, 9.7, 10.2, 11.2-11.6 (broad), 12.5, 13843.9, 14.6.

When propionic anhydride is used in the above experiment in place of acetic anhydride, the sodium-salt of N- propionyl hydroxylamino acetic acid is obtained.

Example 3 A mixture of 600 mg. of hydroxylamino acetic acid and 8 ml. of dichloroacetyl chloride is allowed to stand at about 25 C. for two days. This solution is then evaporated to near dryness in vacuo at 25 C. To the resulting residue of N-dichloroacetyl- N-dichl-oroacetoxyamino acetic acid 10 ml. of water is added and the solution allowed to stand at room temperature for about 10 minutes. It is evaporated in vacuo to near dryness to yield 1.965 g. of wet crystals. 1.043 g. of this material is dissolved in 8 ml. of acetone and the solution diluted with about 12 ml. of petroleum ether (3060 C.). Crystallization begins immediately. 277 mg. of crystalline N-dichloroacetyl hydroxylamino acetic acid are obtained, M.P. 155-156 C. (dec.). The compounds-gives the typical red-purple color. with ferric chloride.

Employing furoyl chloride or )benzoyl chloride inthe above process instead of chloroacetyl chloride yields N A clear solution results.

if uroyl hydroxylamino acetic acid 'and N-benzoyl hy:

droxylamino acetic acid respectively.

' i i Example 4 A. A nutrient medium having the following composition is prepared:

j 4 t t Percent Dextrose 4.0 Cornsteep liquor f 1.0 Hydrolyzed lactalbumin (Edarnin) 2.0 Distilled water 93.0

400 ml. of this medium is added to a 2. liter Erlenmeyer baffled flask and the pH adjusted to 6.8 with 0.1 N sodium hydroxide. It isthen sterilized in an autoclave at 15 pounds per square inch gauge pressure, at 121 C. for 17 28 C. ;At the end of 'this time, the fermentation broth 7 is filtered through a Biichnerfilter funnel precoated with supercel. The filtered fermentation broth containing N formyl hydroxylamino acetic acid inhibitsthe growth of Pinto bean plants, as evidenced by a significant reduction in width of. the'trifoliate leaflets of young Pinto bean width of the leaflets of unsprayed plants.

B. Whena filtered fermentation broth'containing N- formyl hydroxylam-ino acetic acid and produced in the manner described above. is sprayed to run-off on. plots of crab grass, a substantially complete killing of the crab grass is effected over a period of 28 days after spraying. When lawn grasses are similarly treated the growth of such grasses is substantially stopped for a period of about 28 days but grass is not killed.

C. The inoculum for the fermentation described in part A above is prepared in the following manner: A culture of Penicillium purpurrescens (NRRL No. 2819) is grown for 7 days at 28 C. on a slant medium composed of 1.0% dextrose, 0.5% hydrolyzed lactalbumin (Edamin), 1.0% yeast extract, 2.0% agar, and the remainder distilled water. r

To this slant culture is added 10 ml. of the salt solution medium (described by Davis & Mingioli, J. Bact.,' 60

17-28 (1950) without sugar) having the following composition: 7

. Gms.7liter K HPO 7 KIIzPO j 3 Na citrate.2H O .5

1 (NH SO 1,

5 ml. of the resulting spore suspension is added to 50 m1. of a sterilized medium containing 4.0% dextrose, 1.0% cornsteep liquor, 2.0% hydrolyzed, lactalbumin (Edamin), and the remainder distilled water. The pH of this medium is adjusted to 6.8 with sodium hydroxide prior to sterilization. This medium is incubated in a 250 ml. flask at 28 C. for 7 days, the incubation being carried out on a shaker operating at a speed of 220 r.p.m. This broth is employed for the inoculation of the fermentation described in part A above. 7

is prepared and 400 ml. thereof added to each of 43 2 liter baffled Erlenmeyer flasks. The pH of the medium portions is then adjusted to 6.8 with a solution of 0.1 N sodium hydroxide, and each flask sterilized in an auto- 3 clave at 15 pounds per square inch gauge pressure at 121 C. for 17 minutes. After sterilization the flasks are cooled to room temperature.

"Each of the 43 flasks are inoculated with 50 ml. of a growing culture of Per zicillz'um frequentans (NRRL No. 2818). The inoculum is prepared in the same. manner described in Example 4 (c). 7 The inoculated media are incubated on a rotary shaker, the table of which is oper-' ating at 120 r.p.m., for a period of 7 days at a temperatureof 28". C. The resulting broths are filtered through V a supercel coated Buchner filter funnel and the filtered fermentation broths are combined.

, 3 litersof the pooled fermentation broth thus obtained v i are lyophylized. The total yield of solids from this 3 liter portion is 245 g. These solids are triturated with one 1000-ml. and two 700 ml. portions of methanol. The insoluble material is separated by filtration and the methanol solutions combined and evaporated in vacuo to near dryness. This residue thus obtainediis divided into two equalportions and treated as follows:

Portion A is washed with ethanol and the remaining solids dried.- .This material, which weighs about 20 g., is dissolved in. ml. of water and the aqueous solution diluted-with 250ml. of ethanol. Crystallization of the sodium salt of N-formyl hydroxylamino acetic acid begins almost immediately and is essentially complete after Z one hour. The salt is separated by filtration andairdried; yield g.

Portion B is dissolved in 100 ml. of water and ethanol added to the aqueous solution until crystallization of sodiurn N-forrnyl hydroxylamino acetate is complete. The yield of the air-dried salt is 10.8 g. Recrystallization of this material from, aqueous ethanol gives substantially pure material, M.P. 205-210 C. (dec.).

Analysis.-Calcd. for C H NO Na (molecular weight 141.06): C, 25.54; H, 2.86; N, 9.93; Na, 16.3. Found: C, 25.50; H, 2.84; N, 9.91; Na, 16.7.

Example 6 A. 1 g. of monosodium N-formyl hydroxylamino acetate is dissolved in 10 m1. of Water and the solution passed through a column containing ca. 27 ml. of a cation exchange resin of the nuclear sulfonic acid type (Amberlite IR-l) on the hydrogen cycle. The resin is washed with 10 ml. of water and the effluent and wash combined. The total volume is about 20 ml. This is lyophilized and the resulting solids trituratedwith acetone. The acetone solution is evaporated to dryness in vacuo and the solid thus obtained is triturated with ethanol. The ethanol solution is evaporated to dryness in vacuo. The resulting crystalline residue consisting of N-forrnyl hydroxylamino acetic acid is further purified by recrystallization from acetone-petroleum ether (-60 C.). The crystalline (needle-like) N-formyl-N-hydroxylamino acctic acid obtained melts at ll9120 C.

Analysis.-Calcd. for C H NO (molecular weight 119.08): C, 30.26; H, 4.23; N, 11.76. Found: C, 30.88; N, 4.48; N, 12.21.

Potentiometric titration in water with 0.1 N LiOH shows two acidic functional groups: pH /2 ca. 3,5, eq. wt. 123, and pH /2 ca. 9.1, eq. wt. 111.

The acid and its salts have an Rf of 0.3-0.5 in a nbutanolzacetic acidzwater (4:1:1 by volume) system on Whatman No. 1 filter paper.

' B. 269 mg. of monosodium N-formyl hydroxylamino acetate is dissolved in 2 ml. of Water and the solution adjusted to ca. pH l-2 with dilute hydrochloric acid. The solution is evaporated to near dryness in vacuo and the wet residue triturated with three 4 ml. portions of ethanol. The solutions are combined and evaporated to dryness in vacuo. The residue is dissolved in 5 ml. of ethanol and ca. 3 ml. of chloroform added to the solution. A small amount of white precipitate is separated and the clear solution evaporated to dryness in vacuo. The partially crystalline residue of N-formyl hydroxylamino acetic acid is dissolved in ca. 4 ml. of ethanol and the solution diluted with ml. of chloroform. This solution is evaporated to dryness in vacuo to give cube-like crystals of N-formyl hydroxylamine acetic acid, MP. 108- 110 C.

Example 7 5 g. of sodium N-formyl hydroxylamino acetate is dissolved in 20 ml. of water and the resulting solution passed through a column containing 200 ml. of Amberlite IR-120 resin on the hydrogen cycle. When the sodium salt solution has been put over the resin, the resin is then Washed with 59 ml. of Water and this wash combined with the column efiluent. The volume of the resulting solution is about 65 ml. This solution is divided into three portions of 21 ml. each.

One portion is treated with concentrated aqueous ammonium hydroxide to a pH of 8 and the basic solution evaporated to dryness under reduced pressure. The residual mono-ammonium salt of N-formyl hydroxylamino acetic acid is purified by dissolving in 5 ml. of Water and inducing crystallization by addition of ml. of ethanol to the aqueous solution.

The second 21 ml. portion obtained above is treated to a pH of 7-8 with dilute aqueous potassium hydroxide. This solution is then concentrated to dryness and- 8 the residue dissolved in 5 ml. of water. Addition of 35 ml. of ethanol to the aqueous solution results in crystallization of the monopotassium salt of N-forrnyl hydroxylamino acetic acid.

The third portion is treated with excess calcium carbonate. After evolution of carbondioxide is complete, the excess solid calcium carbonate is removed by centrifugation and the clear solution thus obtained concentrated to near dryness. The crystalline material which forms is the calcium salt of N-forrnyl hydroxylamino acetic acid which is only slightly soluble in water.

Example 8 0.5 mg. of N-formyl hydroxylamino acetic acid is dissolved in about 0.5 m1. of dilute aqueous sodium hydroxide. Dilution of the resulting solution with ethanol causes crystallization of the di-sodium solution. Disodium N-formyl hydroxylamino acetate may also be obtained from the monosodium salt by adjusting the pH of a solution containing the mono-salt to about 11-12 with dilute sodium hydroxide. Upon addition to ethanol to this strongly basic solution, the di-sodium salt crystallizes and may be purified by recrystallization from aqueous ethanol.

Potentiometric titration of the di-sodium salt with 0.1 N hydrochloric acid gives two spans--pH /2 ca. 8.7 and pH /2 ca. 2.9.

The di-sodium salt of N-formyl hydroxlamino acetic acid obtained as described above may be converted, if desired, to the monosodium salt by treatment of an aqueous solution to a pH of 6-7 with a small amount of glacial acetic acid. The product is crystallized by dilution of the reaction mixture with ethanol.

Example 9 N-formyl hydroxlyarninoacetic acid is produced in substantial quantities when the process of Example 5 is carried out employing species of Penicillium other than the Penicillium frequentans (NRRL N0. 2818) employed in that example. Species of Penicillium which are particularly useful in elaborating N-forrnyl hydroxylaminoacetic acid are the following:

Penicilliwm" spinulosum, NRRL 2827, Penicillium spinulosum, NRRL 2829, Penicillium frequentans, NRRL 1915, of the Penicillium frequentans series.

Penicilliam trzebinskii, NRRL 732, Penicillium aurantio-violaceum, NRRL 762, Penicillium lividum, NRRL 754, of the Penicillium lividam series.

Penicillium implicatum, NRRL 723, of the Penicillium implicatum series.

Penicilliam turbatum, NRRL 758, of the Penicillium thomii series.

Penicillium janthinellum, NRRL 2828, of the Penicillium janthinellum' series.

Penicilliztm caseicolam, NRRL 876, of the Penicillium camemberti series. I

Penicillium crustosmn, NRRL 967, of the Penicillium expansum series.

Example 10 suspended in mineral oil (Nujol) taken'on a Perkin-Elmer Model 137 Infrared Spectrophotometer (Infracord) shows' characteristic peaks at the following wavelengths, ex-

ether (30-60 C.). The ester '9 pressed in microns: 5.75, 6.0, 7.9, 8.25, 8.45, 9.8, 9.95, 10.35, 111.15, 12.95, 13.3-13.8 (broad), 14.5.

Benzyl N-furoyl hydroxylamino acetate and ethyl-N- phenylacetyl hydroxylamino acetate are prepared by intimately contacting furoyl chloride with benzyl hydroxylamino acetate and phenylacetyl chloride with ethyl hydroxylamino acetate according to the procedure of Example 3 above.

Example 11 Parts per million: Percent kill 4000 99 2000 80 1000 40 Similar results are obtained when the free acid or other alkali and alkaline earth metal salts are used instead of the monosodium salt.

B. A fermentation broth containing N-formyl hydroxylamino acetic acid is produced by fermenting the nutrient medium described in Example 4 above with Penicillium purpurrescens NRRL No. 28-19 by the procedure of Example 4. On completion of the fermentation the solid material is removed by filtering and the filtered broth sprayed to run-off on two separate plots of crab grass and on an established lawn plot in which there were five different lawn grasses; Highland Bent, New Zealand Fescue, Fancy Red Top, Kentucky Blue and Merion Blue. The following table illustrates the effects of spraying the strained fermentation broth on the three plots 28 days after spraying:

Crab grass: 6-9 cm. tall 100% killed.

Crab grass: -20 cm. tall 95% killed.

Lawn grass Growth stopped; grass not killed.

N-formyl hydroxylamino acetic acid, and its salts and esters, are also highly effective in killing sheep sorrel, plantain, dandelions, clover, poison ivy, smart weed and ditch bank weed when aqueous solutions thereof are sprayed to run-oif on the plants. Aqueous solutions of the pure substances or of concentrates such as fermentation solids, or filtered fermentation broths themselves may be employed.

Example 12 The following procedure is used to determine the effectiveness of the compounds of the invention in inhibiting growth of Pinto bean plants:

Two seeds of Pinto bean (Phaseolus vulgaris L.) are planted 1 inch deep in a 3 /2 inch water proof paper pot containing a mixture of 50%sandy loam soil and 50% Michigan hyper humus. After the pot is maintained with normal watering at a temperature of 28 39 C. at a 65% relative humidity in the presence of natural sunlight for one week, the less developed plant is removed from the pot and discarded. The soil of the pot is then fertilized with an aqueous nutrient solution containing calcium nitrate (8.1 gm./l.), potassium nitrate (2.5 gm./l.), potassium dihydrogen phosphate (0.63 gm./l.) and magnesium sulfate (1.2 gm./ 1.). Three days later, when the primary leaves of the remaining plant are about two-thirds of their full size and when the trifoliate leaves are just starting to expand, the entire plant, including both sides of the leaves, is sprayed to run-off with the test solution. A second pot, started at the same time and treated the same way, except that the bean plant is not sprayed, is used as a control. The test plant and the control plant are maintained with Parts per million of monosodium salt: Width of leaflets, mm.

1 B. Three liters of'whole broth produced by fermentation of a nutrient medium with Penicillium purpurrescens (NRRL No. 2819) in a manner similar to that described in Example 4 is acidified to a pH of 4 with acetic acid. The acidified broth is filtered and 1600 ml. of the clear filtrate freeze-dried. Two aqueous solutions containing respectively 10 rug/ml. and 20 mg./ ml. of the freeze-dried solids were tested for inhibition of Pinto bean plant growth by the above procedure. The solution containing 20 mg./ ml. of the freeze-dried solids gave a 92% reduction in width of the trifoliate leaflets as compared with an untreated control plant, and the solution having a 10 mg./ ml.

H of the freeze-dried solids gave a 69% reduction under the same conditions.

Example 13 The tumor-inhibiting properties of the compounds of this invention are demonstrated by use of an embryonated egg test. Nine day embryonated eggs are implanted with pieces of tumor tissue, such as human adeno carcinoma (HAD-1), human bronchiogenic carcinoma (A-42), human epidermoid carcinoma (HEP-3) and human sarcoma (HS-l). The tissue piece is placed on a well-vascularized region of the chlorioallantoic membrane of the eggs, and the eggs are incubated for 3 to 4 days. They are then examined to determine in which eggs the tissue pieces have taken (i.e., started to grow). Such eggs are then divided into two groups: a control group and a group to be treated. Those eggs wherein the tissue has not taken are removed from the test.

The eggs of the group to be treated are injected with 1 ml. of a solution containing one of the N-acyl hydroxylamino acetic acid compounds of this invention (the test solution). The eggs of the control group are injected with 1 ml. of liquid such as Earles saline solution or distilled water. When the eggs are 20 days old, they are harvested by removing the embryos and tumors and weighing them.

The average weight of the embryos treated with test solution is compared with the average weight of the untreated control embryos and the percent retardation (P) of the average weight of the embryos is calculated by means of the formula:

P:(1A/B) wherein A is the average weight of the treated embryos and B is the average weight of the untreated control embryos.

The average weight of the tumors obtained from the treated eggs is compared with the average weight of the tumors from the untreated controls and the percent retardation (Q) of tumor growth is calculated by means of the formula:

wherein C is the average weight of the tumors from the treated eggs and D is the average weight of the tumors from the control eggs.

A. In one experiment employing this test, 1 ml. of Earles saline solution containing 10 mg./ml. of sodium N-formyl hydroxylamino acetate was added to each of 6 test eggs implanted with human adeno carcinoma (HAD-1) tissue. 1 ml. of Earles saline solution containing none of the test compound was added to each of 6 control eggs similarly implanted with the human adeno carcinoma tissue. The test was carried out as described. The sodium N-formyl hydroxylamino acetate caused a 4.5% retardation in average weight of the embryos (P), and a 66% retardation of tumor growth B. When N-formyl hydroxylamino acetic acid was emplayed in place of the sodium salt, a 4% retardation in average embryo weight (P) and a 78% retardation in tumor growth (Q) was observed.

Any departure from the above description which conforms to the present invention is intended to be included within the scope of the claims.

What is claimed is:

1. Compounds of the formula R N-CH2i 3-0R3 where R is selected from the group consisting of formyl, lower alkanoyl, haloloweralkanoyl, furoyl, homocyclic aroyl, and aralkanoyl, R is selected from the group consisting of hydrogen, an alkali metal, an alkaline earth metal and lower alkanoyl and R is selected from the group consisting of hydrogen, ammonium, an alkali metal, an alkaline earth metal, lower alkyl and aralkyl.

2. N-formyl hydroxylamino acetic acid.

3. N-acetyl hydroxylamino acetic acid.

4. N-benzoyl hydroxylamino acetic acid. 5. An alkali metal salt of N-formyl hydroxylamino acetic acid.

6. Sodium N-formyl hydroxylamino acetate.

7. Lower alkyl N-formyl hydroxylamino acetate.

8. Potassium-N-formyl hydroxylamino acetate.

9. Methyl-N-formyl hydroxylamino acetate.

10. Ethyl-N-formyl hydroxylamino acetate.

References Cited by the Examiner UNITED STATES PATENTS 2,847,455 8/58 Steadman et al. 260482 2,880,232 3/59 Frankel et al. 250-482 2,947,665 8/60 Foster 36 2,949,404 8/60 Sakaguchi et al. 19536 OTHER REFERENCES Sidgwick, The Organic Chemistry of Nitrogen, pages 197-201 (1945).

Wieland et al., Chem. Ber., 89, 2408-2414 (1956).

LORRAINE A. WEINBERGER, Primary Examiner.

ABRAHAM H. WINKELSTEIN, LEON ZITVER,

Examiners. 

1. COMPOUNDS OF THE FORMULA 